Preserved, non-infectious control cells prepared by the modulation or modification of normal cells

ABSTRACT

The invention describes the preparation of preserved, non-infectious control cells. The control cells are prepared by modification, activation or otherwise changing selected normal leukocyte cells, tissue culture cells or zenogenic transplants in immunosuppressed animals to have characteristics of abnormal cells or by the addition of cells from an established cell line, which has been similarly modified or activated, to a normal leukocyte population. The preferred method of preserving the control cells is by lyophilization using an isotonic 10% trehalose solution.

RELATED INVENTION

This application is related to U.S. Pat. No. 5,059,518, issued Oct. 22,1991 and to application Ser. No. 07/944,678, filed Sep. 14, 1992, andentitled PRESERVED, NON-INFECTIOUS CONTROL CELLS FOR USE IN THEIDENTIFICATION OF A DISEASE THROUGH BLOOD TESTING. This application,U.S. Pat. No. 5,059,518 and application 07/994,678, now abandoned, areowned by a common assignee, Coulter Corporation, Hialeah, Fla.

FIELD OF THE INVENTION

This invention relates to control cells and a method of producing thesame for use in clinical and immunological analytical procedures.Specifically, this invention relates to preserved, non-infectiouscontrol cells which are derived from peripheral blood cells or anestablished cell line. The cells are modified or changed to have thecharacteristics of abnormal cells. These control cells have, among thetotal population or count of cells in a given control cell sample, apopulation or count of at least one specific cell type which has anamount or degree of antigenic (antibody-binding) expression, orexpression of cellular constituents (for example, enzymes or nucleicacids) that is characteristic of:

(1) abnormal peripheral blood cells obtained from a

patient suffering from a disease, or

(2) normal hematologic tissue different from normal peripheral bloodcells, such as, bone marrow cells and the cells derived therefrom, forexample, red blood cells.

BACKGROUND OF THE INVENTION

Control cells are essential for the accuracy and precision of clinicaland immunological assays. They are needed to insure the reliability andaccuracy of test equipment and methods and to insure reproducibilitythrough time and from laboratory to laboratory. State and federalregulations which govern such assays often require the use ofmulti-level controls in order to demonstrate that equipment isperforming properly over a range of values. In immunological assays,fresh normal cells have been the standard control cells for suchequipment testing. In order to avoid the cost and expense of obtainingand maintaining fresh cells, various methods of preserving fresh cellsfor use as control cells have been evaluated. For example, U.S. Pat. No.5,059,518 (the '518 patent) describes the use of lyophilized normalcells for use as control cells.

Abnormal blood cell samples have also been used as controls to confirmthe presence of a disease or to determine its stage, but their use hasbeen restricted to fresh or refrigerated samples obtained from diseasedpatients. The supply of such samples is thus inherently restricted,inherently variable and of limited shelf life. Furthermore, since manyof such blood samples are also associated with infectious diseases, theyare not amenable, for health and social as well as sometimes technicalreasons, to large scale production and distribution, and they requirespecial handling procedures.

Some aspects of the problem may be overcome as disclosed in relatedapplication Ser. No. 07/944,678 which describes the use of control cellswhich reflect the abnormal extant state of a blood cell populationarising from:

(1) an increase or decrease in the number of cells of a specific typedue to the presence of a disease; or

(2) the presence of cells not normally found in normal blood samples,such cells being different in size, shape or other physicalcharacteristics, or having thereon molecules and/or antigenic sites notfound on the normal cells.

The control cells of the first type are prepared by depleting a bloodsample of specific cells normally present in blood to less than normallevels or by adding such cells to above normal levels and subsequentlypreserving such samples. The control cells of the second type areprepared by the addition of cells not normally present in a normal bloodsample to a normal blood sample and subsequently preserving such sample.

This invention describes a method of preparing, from normal cells or acombination of normal cells and a selected, established cell line,control cells which can be used as an alternative to abnormal cellsobtained from patients. To make the control cells of the invention,normal cells obtained from disease free patients or cultured cells areobtained from an established cell line. These cells are subsequentlymodified, altered or changed to so that they reflect the characteristicsof one or more specific cells types which would be present in the bloodof a patient having a specific disorder or disease. The normal orcultured cells can also be modified to reflect the characteristics ofcertain cell types found in tissues as opposed to peripheral blood. Forexample, normal cells can be modified to reflect the antigeniccharacteristics of bone marrow cells and derivatives thereof.

The modulation or modification of cells to produce appropriate controlcells is not limited to the expression of any one particular cellularconstituent and represents a cascade of events reflected in changes inmany constituents. Thus the modulation or modification of cells isperformed to produce control cells that are appropriate for theconstituent(s) being analyzed--be they antigenic constituents as testedby monoclonal antibody binding, enzymatic assays as determined bysubstrate modification or nucleic acid changes as demonstrated by DNA orRNA probes. The antigenically defined components are, therefore, theendpoint of a cascade of cellular events and the product of DNA/RNAdirected biosynthesis.

SUMMARY OF THE INVENTION

The invention provides for mammalian cells which reflect a specific cellpopulation in a blood or tissue sample of a mammal which has an disease,disorder or condition which manifests itself, among other ways, as achange in the expression in type, number or physicochemical propertiesof components present on or in cells. Antigenically defined componentsare antibody binding sites. Other components may consist of enzymes, andtheir active nucleic acid containing materials such as DNA or RNA,cellular macrostructures such as ribosomes and the configuration ofnuclear components such as the nucleus. The invention further providesmethods for producing such control cells.

The control cells described herein are produced from normal,non-infectious, non-disease-altered peripheral blood cells or from anestablished cell line by the use of known means to activate or modifythe cell's own biosynthesis mechanisms. The control cells also can beproduced by chemically or enzymatically modifying specific chemicalgroups on a cell or by inhibiting the expression of antigenicallydefined characteristics. The altered cells can be added to normal cellsin selected amounts. Different amounts of specific cells in theperipheral blood of tissue sample is often reflective of the stage of adisease (how far it has progressed) or disorder (an abnormal physicalcondition).

The control cells are prepared by activating or inhibiting the cells ownbiosynthetic mechanisms, or chemically or enzymatically modifying theantigenically defined components in or on normal peripheral blood cellsor cells from an established cell line; or by growing the cells in animmunosuppressed laboratory animal in which various biochemical reagentsare used to modify cell growth. The normal blood cells or cells from anestablished cell line that have been so activated, modified, inhibitedor grown can be preserved, for example, by lyophilization, so as to havean extended storage life. The control cells which are prepared andpreserved according to the invention retain, after reconstitution, allthe antigenicity and cell surface structural characteristics andcellular constituents of the corresponding fresh cells.

The expression of antigenically defined components is known to be areflection of cellular DNA and RNA in association with the cell'sbiosynthetic structures. The detection of antigenically definedstructures is the detection of the final products of a cascade ofevents. Detection is feasible at various stages in this process--be itDNA or RNA changes, or an alteration in various cellular enzymes orphysical structures. Detection may be done using monoclonal antibodies,DNA and RNA probes, enzymes and other biological and chemical substanceknown to these skilled in the art.

The invention also provides for a process for preparing the preservedmammalian control cells. In the process selected leukocyte cells, tissueculture cells or zenogenic transplants in immunosuppressed animals areartificially activated and/or modified to express a characteristicexpressed on similar cells in a person having a disease or disorderwhich results in the expression of the characteristics on the cells. Themethod comprises, for example using leukocyte cells, incubating a sampleof normal leukocyte cells in a medium, typically a fluid or solution ofsuitable composition for the incubation, and in the presence of anenzyme or chemical reagent to selectively activate or modify theantigenic characteristics of a selected population of the leukocytecells such that the selected population of cells has characteristicsrepresentative of the same cells in a patient suffering from a diseaseor disorder which results in the expression of such characteristics onthe selected cells. The treated leukocyte cells are then separated fromthe medium and washed. After washing, the cells are preserved by asuitable preservation technique. The preferred preservation technique islyophilization after suspension in an isotonic 10% trehalose solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a histogram illustrating the distribution of normal peripheralblood lymphocytes activated with 3 μg/ml PHA for 36 hours.

FIG. 2 is a histogram illustrating the distribution of normal peripheralblood lymphocytes cultured for 96 hours without PHA.

FIG. 3 is a histogram illustrating the distribution of normal peripheralblood lymphocytes activated with μg/ml PHA for 36 hours, lyophilized andreconstituted before use.

FIG. 4 illustrates the distribution and count of CD34 positiveleukocytes in the peripheral blood of a patient with acute myelogenousleukemia (AML).

FIG. 5 illustrates the distribution and count of CD34 positive bonemarrow cells in a normal bone marrow sample.

FIG. 6 illustrates the distribution and count of lyophilized CD34positive KG1a tissue culture cells mixed with normal peripheral bloodlymphocytes.

FIG. 7 illustrates the distribution and count of reconstituted,lyophilized, chemically modified CD34 positive KG1a tissue culture cellsmixed with normal peripheral blood lymphocytes.

FIG. 8 is a second illustration of the distribution and count ofreconstituted chemically modified CD34 positive KG1a tissue cells mixedwith normal peripheral blood lymphocytes.

FIG. 9 is an illustration using PHA activated peripheral bloodlymphocytes and demonstrating in situ hybridization for the mRNAassociated with the cell's production of Interleukin-2.

FIG. 10 is an illustration using PHA activated peripheral bloodlymphocytes and demonstrating in situ hybridization for the mRNAassociated with the cells' production of ICAM-1.

FIG. 11 is a flow chart illustrating the process of modulation ormodification of cells for use as abnormal control cells.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "antigen modified control cell" signifies a control cell inwhich the expression of an antigen in a specific cell type or typesdiffers from that in cells found in a normal blood sample or asexpressed in cells found in hematological tissue other than normalblood.

The term "abnormal blood sample" refers to a blood sample taken from apatient suffering from a disease or disorder. The term "abnormal bloodcells" or "abnormal cells" refers to those cells which exhibitantigenicity or structural characteristics different from normal cellsas a result of a patient having a disease or disorder.

The terms "activated" or "artificially activated", "modified" or"artificially modified", "inhibited" or "artificially inhibited", and"altered" or "artificially altered", and the use of the word"artificial" in conjunction with another word signifying change, areused herein to distinguish cells which have altered characteristics,antigenically or otherwise as compared to normal cells, as a result ofthe use of chemical reagents, enzymes, radiation and other reagentseither taught herein or known to those skilled in the art as effectingsuch changes. These cells are thus distinguished from cells which havethe same characteristics due to their being obtained from a patient whohas a disease or disorder which result in similar characteristics beingpresent on cells.

All solution concentrations given herein are in weight percent unlessspecified otherwise.

Description

The claimed invention uses various means to activate, inhibit, or modifynormal cells or cells cultured from an established cell line. Some ofthese methods have been described in the scientific and technicalliterature. W. Knapp et al., in a publication appearing in LEUKOCYTETYPING IV, H. Stein and R. Schwarting, Eds. (Oxford University Press1989), pages 384-513, report that processes which stimulate cells toproliferate results in a change in the expression of cellular antigenson those cells. D.T.Y. Yu et al., in a publication entitled "Peripheralblood Ia-positive T cells: Increases in certain diseases afterimmunization", J. Exp. Med. 151: 91-100 (1980), report that theactivation of T lymphocytes causes an increase in the expression of typeII MHC antigens on the T cells as would be seen on the cells of patientswho have certain diseases or after patient immunization. Activation ofnormal peripheral blood T cells by known means such as certain plantlectins (PHA, PWA) crosslinked antibodies (CD3, CD11, CD28 ) modulatescellular antigens. That is, it increases the number of cells having thetype II MHC antigens. As a result, cells which have been activated ormodified to exhibit the MHC antigens can mimic the type of cells foundin clinical situations. The activated or modified cells can be preservedand used as control cells for clinical and immunological analyticalprocedures.

C. I. Civing et al. report in LEUKOCYTE TYPING IV, op. cit., page 818,that in some patients suffering from acute myelogenous leukemia (AML), aperipheral blood sample will contain cells which have a CD34 antigen notpresent on normal blood cells. The enumeration of CD34 positive cells inbone marrow samples is used in bone marrow transplantation procedures toevaluate precursor cells populations. See BONE MARROW PROCESSING ANDPURGING: A PRACTICAL GUIDE, A. P. Gee, Ed. (CRC Press, Boca Raton,Fla.), pages 145-148. The expression of CD34 on these cells is variableand is usually less than that seen in tissue culture cells, for example,KG1a cells, available from the American Type Culture Collection (ATCC),Rockville, Md., ATCC Deposit No. CCL 246.1. Modification of the CD34antigen on cultured cells by chemical or enzymatic means can providecontrol cells which mimic the cellular expression of the antigen as seenunder a variety of clinical conditions.

In order to identify a particular type of cell associated with aspecific disease or condition, several different molecular structuresmay have to be identified on a single cell. A broad class of cells mayhave to be increasingly subdivided in to smaller and more specificsubsets. In order to distinguish one broad class of cells within severalbroad classes of cells, and to further distinguish subsets within theselected broad class of cells, current technology uses selectedmonoclonal antibodies bearing different labels. For example, one mayuse, alone or in combination, labelling molecules which containradioactive elements, enzymes and dyes as the labels. Dyes, especiallyfluorescent dyes, are preferred. Examples of such fluorescent dyesinclude fluorescein, fluorescein isothiocyanate (FITC),tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE or RD),phycoerythrin-Texas Red conjugates and allophycocyanin.

The application of in situ DNA and RNA hybridization techniques can beused to identify particular cellular characteristics in a similarfashion. In this case, the labelled probes are used to detect theRNA/DNA components associated with a specific disease or condition. G.J. Bauman et al., "Fluorescent In-Situ Hybridization To Detect CellularRNA By Flow Cytometry And Confocal Microscopy", J. Microscopy 157 (Part1): 73-81 (January, 1990). "Fluorescent In Situ Hybridization ToInterphase Cell Nuclei In Suspension Allows Flow Cytometric Analysis OfChromosome Content And Microscopic Analysis Of Nuclear Organization", B.Task et al., Human Genetics 78:251-259 (1988). DNA or RNA probes aregenerally a short oligonucleotide chain complimentary to a chainoccurring in the molecule or cell of interest. For example, a DNA probeof sequence GGTATACC would be complimentary to one present in a cellhaving the sequence CCATATGG.

The control cells expressing a specifically modified antigen can beprepared from leukocyte-rich, anti-coagulated blood or from a selectedtype of cultured cells. In the first instance, a sample ofleukocyte-rich, anti-coagulated blood is lysed of red cells (RBCs) andthe lysis debris removed by washing. Alternatively, red blood cells maybe removed by other suitable means such as the use of anerythrocyte-specific antibody conjugated to an insoluble substrate asdescribed U.S. Pat. No. 4,752,563. If desired, specific types ofnon-erythrocyte cells can also be removed from the blood sample by theuse of additional selective antibodies conjugated to insoluble supports.For example, a monoclonal antibody specific to an antigen present onlyon granulocytes could be conjugated to an insoluble support and theresulting conjugated antibody used to remove granulocytes from anerythrocyte-free blood sample. Glass, ceramic or polymeric beads, eithermagnetic or non-magnetic, are among the many types of supports which maybe used. Selected cells may also be removed by the use of well knowndensity gradient techniques.

Selected cells can be removed from the original blood sample and theseremoved cells or the cells remaining in the sample may be activated bymeans known in the art. For example, some methods of activating cells isdiscussed in CURRENT PROTOCOLS IN IMMUNOLOGY, J. Coligan, A. Kruisbeek,D. Margulies, E. Shevach and W. Strober, Eds. (Greene PublishingAssociated and Wiley-Interscience, New York 1991), Section 3.12 entitled"Proliferative Assays for T Cell Function". After the cells have beenactivated, they are preserved by the procedures described in U.S. Pat.No. 5,059,518, whose teachings are incorporated herein by reference, orby other suitable preservation techniques known in the art. Theprinciple criterion in the choice of the preservation technique is thatthe preserved cells, when reconstituted or otherwise readied for actualuse in an assay, have retained the antigenicity or other test-relevantcharacteristics the cells had prior to preservation. The control cellsprepared according to this method have present a spectrum of leukocytecells which exhibit the expression of cellular constituents, includingantigenic components, enzyme activity or nucleic acid expression, thatis characteristic of and similar to those of leukocyte cells naturallyactivated in a patient suffering from a disease or disorder. The controlcells so prepared are thus suitable for use in clinical andimmunological assays which measure leukocyte activation and rely on thecharacteristics.

Cultured cells from a selected, established cell line can also be usedto prepared control cells according to the invention. Cells of the cellline are grown an tissue culture medium using established means,harvested and further processed using chemical or enzymatic proceduresto modify the cells' antigenic structure(s). For example, KG1a cells areharvested and treated with iodoactamide to modify the CD34 antigenexpression. The modified cells are then added to a leukocyte samplewhich was produced from leukocyte-rich, anti-coagulated blood from whichthe RBCs and, if desired, other selected cells have been removed by thesame techniques as described above. The resulting cell sample may bepreserved for use as control cells according to U.S. Pat. No. 5,059,518or other suitable preservation techniques. The preserved samplecontaining the modified KG1a cells present a spectrum of leukocyte cellshaving antigenic modifications similar to those that occur in peripheralblood or bone marrow. When reconstituted or otherwise prepared for use,they are suitable for use as control cells in clinical and immunologicalassays.

The invention may be carried out in many different forms. That is, manydifferent types of cells from different mammalian species may beprepared for use as control cells. The invention thus has veterinary aswell as human applications. The examples given herein are to illustratethe teachings and practice of the invention and are not to be taken as alimitation on the scope of the invention. Therefore, while the examplesherein provide for use of human T cells to illustrate the invention,non-T cells or cells from other mammalian species also may be used toprepare control cells useful in the analysis of different diseases indifferent species.

FIG. 11 is a flow chart illustrating the various modifications ormodulations done to cells prior to their being used as control cells,with or without mixing with other cells, for example, normal cells.Following one path, left-to-right from upper left, normal blood cells,tissue culture cells or zenogenic transplants from immunosuppressedanimals are treated with bioregulatory agents. The resulting cells maybe either preserved and subsequently used as control cells or they maybe further processed using chemical agents before preservation. In thesecond path, top-to-bottom from upper left, the exemplary cells are onlymodified before preservation and subsequent use as control cells.

EXAMPLE 1 Control Cells Suitable For Use In An Analysis Of T CellActivation

T cell activation is an indication of a variety of clinical conditions.The measurement of Type II MHC (DR) antigen is used to determine T cellactivation. Preserved, non-infectious control cells containing activatedT cells are shown to be useful in an assay measuring T cell activation.

Type II MHC (DR) antigen-modified T cells were prepared from normal,anticoagulated, leukocyte-rich peripheral blood. Several normal,leukocyte-rich blood samples were pooled and the lymphocytes isolatedusing the density gradient technique in which the blood is diluted withphosphate buffered saline (PBS), layered over Ficoll-Hypaque®(Pharmacia, Piscataway, N.J.) and centrifuged. Lymphocytes in theinterface were removed and washed with in PBS. The washed lymphocyteswere then treated with 3 μg/ml phytohemagglutinin (PHA, DIFCO Inc.) andincubated in supplemented RPMI media for about 72-96 hours. At the endof the incubation period, the cells were harvested, washed with PBS oran isotonic 10% trehalose solution, suspended in an isotonic 10%trehalose solution and lyophilized according to U.S. Pat. No. 5,059,518.The preserved control cells, after being reconstituted, were evaluatedby comparison with fresh cells. The results are shown in FIGS. 1-3. Inthese figures, LFL1 represents the fluorescence from an anti-CD3-FITCmonoclonal antibody and LFL2 represents the fluorescence from ananti-DR-RD1 monoclonal antibody. Both are obtainable from CoulterCorporation, Hialeah, Fla.

FIG. 1 is a histogram obtained using fresh, that is non-preserved, PHAtreated normal peripheral blood lymphocytes. FIG. 2 is a histogram offresh lymphocytes that were not treated with PHA. FIG. 3 is a histogramof normal peripheral blood lymphocytes treated with PHA (3 μg/ml),lyophilized and reconstituted prior to use as control cells.

FIGS. 1 and 2 demonstrate the increase in the expression of Type II MHCantigen on T cells which have been activated with PHA (FIG. 1, Quad 2)as compared to T cells that have not been activated (FIG. 2, Quad 2).When FIG. 3 is compared with FIGS. 1 and 2, is evident that the controlcells, activated and preserved as taught herein show, the same increasein MHC antigen expression (see Quad 2) as seen in FIG. 1, Quad 2.

FIGS. 9 and 10 demonstrate the use of labelled oligonucleotide probes todetect changes in cellular mRNA that are associated with cellactivation. FIG. 9 is a histogram obtained using fresh (that is,non-preserved) PHA treated normal peripheral blood lymphocytes fixed in20% acetic-acid, 75% ethanol, 5% water solution for 10 minutes. Thefixed cells are then centrifuged at 700 rpm for seven minutes, separatedfrom the supernatant liquid and resuspended in SSC solution (0.15M NaCl,0.015M sodium citrate, pH 7.0, hereinafter referred to a 1×SCC). Amixture of six biotin labelled anti-sense region specificoligonucleotide probes for human Interleukin-2 mRNA was purchased fromBritish Bio-technology Products Ltd., 4-10 V-Quadrant, Barton Lane,Adington, Oxon OX14 3YS, United Kingdom. Procedures and methods wereaccording to the manufacturer's instruction except that the washsolution used in this procedure comprised 0.4M guanidine thiocyanate,0.1×SSC solution (the standard 1×solution described above was diluted toone (1) part in 10 ) and 0.1% Triton X-100. Peak A in FIG. 9 is the flowcytometric histogram of normal PBLs. Peak B demonstrates the increase inlabelling cells with the Interleukin-2 probes after activation.

FIG. 10 is a flow cytometric histogram obtained in the same manner asthe histogram of FIG. 9 except that the region specific oligonucleotideprobe cocktail was for human ICAM-1 mRNA. The probes are available fromBritish BioTechnology. Peak A in the histogram shows normal PBLs andPeak B demonstrates the increase in labelling with the ICAM-1 probesafter activation.

The following protocol was used for solution hybridization and flowcytometry. These instructions generally follow those supplied by BritishBio-technology for the probes described above.

Protocol For Solution Hybridization And Flow Cytometry

A. DEPC-Water Preparation.

Sufficient diethylpyrocarbonate (DEPC) is added to distilled water togive a final concentration of 0.1% . The mixture is shaken well, allowedto stand overnight and autoclaved at sterilizing temperatures and cooledbefore use. All water used to make the solutions used in this procedurewas similarly autoclaved prior to use.

B. Cell Fixation.

1. Dislodge cells and prepare a cell suspension.

2. Wash the suspension with isotonic saline containing 10% trehalose(U.S. Pat. No. 5,059,318, incorporated by reference).

3. Centrifuge at 700 rpm for 7 minutes.

4. Remove supernatant and discard.

5. Suspend the cells in 10 ml of a solution comprising 10% acetic acid,40% ethanol and 50% water for 10 minutes at room temperature (15°-27°C.). (Note: Other fixing solutions such as the 20% acetic acid, 75%ethanol and 5% water solution described above may also be used.)

6. Centrifuge at 700 rpm for 7 minutes at room temperature.

7. Re-suspend cells in 1 ml of 2×SCC buffer (twice concentration of1×SCC).

C. Hybridization.

1. Use 0.1 ml of cell suspension, containing 1-50×10⁶ cells, for eachhybridization.

2. Spin cells to form a pellet and remove supernatant liquid.

3. Re-suspend cells in 50 μL of hybridization cocktail that has beendenatured by heating for 5 minutes at 95° C. The hybridization cocktailcomprises:

50% Formamide

600 mM NaCl

Sheared Salmon Sperm DNA (prepared by adding 100 mg of salmon sperm DNAto 10 ml of sterile distilled water and letting the sperm DNA dissolveovernight at 4° C. Shearing is done by sonication).

1×PE solution (prepared by dilution of a 10×PE solution that is preparedby dissolving the following at 65° C.).

    ______________________________________                                        (a) Tris-HCL, pH 7.5  0.5      M                                              (b) Sodium pyrophosphate                                                                            1%       w/v                                            (c) Polyvinylpyrolidone (MW 40K)                                                                    2%       w/v                                            (d) Ficoll (MW 400K)  2%       w/v                                            (e) EDTA (disodium)   50       mM                                             ______________________________________                                    

4. Hold the suspension of 3 for 2 hours at 42° C.

5. Place the suspension in a 50 ml conical table and add 25 ml of asolution containing 0.4M guanidine thiocyonate, 1×SSC and 0.1% TritonX-100.

6. Centrifuge for 10 minutes at 2500 rpm.

7. Remove the supernatant liquid and add 25 ml of a solution comprising0.1×SSC and 0.1% Triton X-100 to resuspend the cells.

8. Centrifuge for 10 minutes at 2500 rpm and remove the supernatantliquid, incubate with 1:1,000 Avidin-FITC (Veclor, USA) for 15 minutesat 22° C. Add 1 ml Isoton III (Coulter, Miami, Fla.), centrifuge for 10minutes at 2500 rpm and remove the supernatant liquid.

9. Resuspend the cells in 0.3 ml of Isoton III or equivalent for use inthe flow cytometer.

EXAMPLE 2 Control Cells Suitable For Use In An Analysis of CD34 PositiveCells

The CD34 positive cells of normal, non-diseased donors are associatedwith levels of hematopoietic progenitor cells and are related to thefunctioning of bone marrow cells. However, the low frequency (number orcount) of these cells in a normal blood sample makes their detection andenumeration difficult. Some diseases manifest themselves early-on by achange in the level of CD34 positive cells. Consequently, it is desirousto have a available control cells which aid in the confirming thedetection of CD34 positive cells and in their enumeration.

CD34 positive cells were obtained from the ATCC in the form of a sampleof Deposit No. CCL246 1, the KG1 a cell line. The KG1a cells of thesample were cultured, harvested and placed in a chemical modifyingmedium (CMM) comprising of an isotonic solution containing iodoacetamide(10 mg per 50 ml), levanisole (200 ml per liter), phenylmethylsulfonylfluoride (1 μg/ml) and 0.1% sodium azide. The cells were then incubatedfor a time in the range of about 1-5 hours, and preferably about 1-3hours. The degree of chemical modification to the KGla cell line isdirectly proportional to length of the incubation period.. Afterincubation, the cells were washed once with isotonic 10% trehalosesolution and placed in 0.5% paraformaldehyde solution for about 12hours. The chemically modified KG1a cells were isolated from theparaformaldehyde solution by centrifuging and were suspended in isotonic10% trehalose solution for use in the next step.

In the next step, non-infectious leukocytes were prepared by firstpooling a plurality of normal, anti-coagulated leukocyte-rich bloodpacks and lysing the red blood cells. After lysis, the leukocyte cellswere washed a plurality of times with Hepes buffered saline (HBS). Theleukocyte cells were then separated from the final wash and placed in avessel with isotonic 10% trehalose solution. The chemically modifiedKG1a cells were then added to noninfectious normal leukocytes andresulting mixture lyophilized to produce preserved control cellssuitable for use in a CD34 assay. Before use in such an assay thecontrol cells were reconstituted in distilled water. The control cellswere evaluated flow cytometrically using fluorescently labelledmonoclonal antibodies. FIGS. 4-8 represent a comparison of analyticalresults obtainable using control cells prepared and preserved accordingto the invention and fresh CD34 positive cells obtained from a patient.

FIG. 4 depicts the CD34 positive leukocytes in the peripheral blood of apatient suffering from AML. LFL2 represents the fluorescence from cellsconjugated to an anti-CD34-PE monoclonal antibody (Becton-DickinsonImmunology Systems, San Jose, Calif.). CD34 positive cells above thebackground level are indicated by histogram gate No. 1.

FIG. 5 depicts the CD34 positive cells in a normal bone marrow sample.The x-axis of the histogram represents the fluorescence from cellsconjugated to an anti-CD34-RD1 monoclonal antibody (CoulterCorporation). Based on isotype control, histogram gate "C" indicates thepositive cells above background level.

FIG. 6 depicts reconstituted lyophilized control cells consisting ofCD34 positive KG1a tissue culture cells that have been mixed with normalperipheral blood lymphocytes prior to lyophilization. LFL2 representsthe fluorescence from cells conjugated with an anti-CD34-PE monoclonalantibody (Becton-Dickinson). CD34 positive cells above the backgroundlevel are indicated by histogram gate number 1. The level offluorescence indicated by "mean" represents the degree of expression ofthe anti-CD34 monoclonal antibody binding capacity.

FIG. 7 depicts reconstituted lyophilized control cells consisting ofchemically modified CD34 positive KG1a tissue culture cells that weremixed with normal peripheral blood lymphocytes before lyophilization.LFL2 represents the fluorescence of those cells conjugated to ananti-CD34-PE monoclonal antibody (Becton-Dickinson). CD34 positive cellsabove the background level are indicated by histogram gate number 1. Thelevel of fluorescence indicated by the "mean" channel (155) presents thereduced degree of expression of the anti-CD34 monoclonal antibodybinding capacity that occurs after chemical modification.

FIG. 8 depicts reconstituted lyophilized control cells consisting ofchemically modified CD34 positive KG1a tissue culture cells mixed withnormal peripheral blood lymphocytes prior to lyophilization. LFL2represents the fluorescence of those cells that are conjugated to ananti-CD34-PE monoclonal antibody (Becton-Dickinson). CD34 positive cellsabove the background level are indicated by histogram gate 1. The levelof fluorescence indicated by the "mean" channel represents a furtherreduction of the degree of expression of anti-CD34 monoclonal antibodybinding after increased chemical modification by extending theincubation period over that for the sample of FIG. 7.

FIGS. 6-8 compare normal lymphocytes which have been augmented withnon-modified CD34 positive KG1a cells (FIG. 6) with normal lymphocytesthat have been augmented with KG1a cells that have chemically modifiedto express different degrees of CD34 antigen expression. The results inFIG. 6 represent the baseline. That is, if the percentage of KG1a cellsis constant in the samples represented by FIGS. 6-8, the number of cellsexpressing the CD34 antigen will be greatest in FIG. 6. Chemicalmodification of the KG1a cells destroys the expression of the CD34antigen as indicated by a reduction in mean Tables 6-8. FIGS. 7-8represent samples with the reduced expression.

The results shown herein by FIGS. 1-8 indicate that the method of theinvention can be used to prepare lyophilized, non-infectious controlcells which contain differing levels of CD34 expression and that thesecontrol cells, when reconstituted, can be used in clinical andimmunological assays.

The following data tables correspond to the indicated figures.

                  TABLE 1                                                         ______________________________________                                        FIG. 1.                                                                                                PER-              %                                  MIN     MAX     COUNT    CENT  MEAN   SD   HPCV                               ______________________________________                                        1 X  0      10       260    5.2   4.3   3.5                                   Y    7      63                   36.6   16.8  1.54                            2 X  11     63      2943   58.9  29.3   5.9  13.9                             Y    7      63                   25.6   12.5 37.2                             3 X  0      10       177    3.5   2.3   3.5                                   Y    0       6                    1.0   1.8                                   4 X  11     63      1619   32.4  29.5   5.6  10.9                              Y   0       6                    1.1   1.9                                   ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        FIG. 2.                                                                                                PER-              %                                  MIN     MAX     COUNT    CENT  MEAN   SD   HPCV                               ______________________________________                                        1 X  0       2      188    3.8    0.1   0.4                                   Y    9      63                   36.6   11.1 5.75                             2 X  3      63      200    4.0   28.1   6.7  7.41                             Y    9      63                   20.1   12.2 14.7                             3 X  0       2      365    7.3    0.0   0.2                                   Y    0       8                    1.1   2.0                                   4 X  3      63      4246   84.9  29.5   3.4  8.14                             Y    0       8                    1.0   2.0                                   ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        FIG. 3                                                                                                 PER-              %                                  MIN     MAX     COUNT    CENT  MEAN   SD   HPCV                               ______________________________________                                        1 X   0     13       432    8.6   5.7   4.7                                   Y    19     63                   50.1   10.3                                  2 X  14     63      1255   25.1  31.1   6.6  10.1                             Y    19     63                   36.4   12.2 32.5                             3 X   0     13       260    5.2   5.1   4.8                                   Y     0     18                    3.7   4.9                                   4 X  14     63      3052   61.1  32.2   5.3  12.3                             Y     0     18                    4.0   4.7                                   ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        FIG. 4                                                                                                 PER-              %                                  MIN     MAX     COUNT    CENT  MEAN   SD   HPCV                               ______________________________________                                        1    7.960  1023.   6991   69.9  14.95  1.47 12.6                             ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        FIG. 6                                                                                                 PER-              %                                  MIN     MAX     COUNT    CENT  MEAN   SD   HPCV                               ______________________________________                                        1    78     255     1176   33.6  206.8  25.0 4.22                             ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        FIG. 7                                                                                                 PER-              %                                  MIN     MAX     COUNT    CENT  MEAN   SD   HPCV                               ______________________________________                                        1    65     255     574    16.4  155.2  34.0 0.85                             ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        FIG. 8                                                                                                 PER-              %                                  MIN     MAX     COUNT    CENT  MEAN   SD   HPCV                               ______________________________________                                        1    72     255     820    23.4  99.4   14.8 1.40                             ______________________________________                                    

We claim:
 1. Control cells composition for use in clinical andimmunological assays, utilizing flow cytometry comprising a preservedmixture of (a) normal mammalian leukocyte cells and (b) selected normalmammalian leukocyte cells which have been artificially enzymatically orchemically activated or modified to express antigenic characteristicsexpressed on the same leukocyte cells in a person having a disease ordisorder which results in the expression of said characteristics on saidselected leukocyte cells; said mixture is preserved by lyophilizationwith an isotonic trehalose solution.
 2. The control cell composition ofclaim 1 wherein the ac-tivated or modified leukocyte cells of part (b)consist of a subset of the normal leukocyte cells of part (a) which havebeen activated or modified.
 3. The control cell composition according toclaim 1 wherein said activated or modified cells are T and B cells. 4.The control cell composition according to claim 1 wherein said activatedor modified cells are T cells.
 5. The control cell composition accordingto claim 1 wherein said cells are suitable for replacing fresh abnormalmammalian cells as standards in cell flow cytometric analysis. 6.Control cell composition for clinical and immunological flow cytometryassays comprising a mixture preserved with isotonic trehalose of thefollowing which are suitable for use in flow cytometry assays:(a) normalmammalian leukocyte cells; and (b) normal mammalian cells which havebeen artificially inhibited during an incubation period, of modified, oractivated, so as to possess antigenic characteristics expressed on thesame type of cells in a person having a disease of disorder whichresults in the expression of said characteristics on said cells.
 7. Thecontrol cell composition of claim 6 wherein the cells of part (b) are Tcells, B cells and tissue cells.
 8. A process for preparing a mixture ofpreserved mammalian control cells useful in flow cytometry analysis inwhich a selected population of leukocyte cells in a normal leukocytecell population have been artificially activated or modified to expressantigenic characteristics which are expressed on the same leukocytecells in a person having a disease or disorder, said processcomprising:(a) incubating a sample of normal leukocyte cells in a mediumwith an enzyme or chemical reagent in order to selectively activate ormodify the antigenic characteristics of a selected population ofleukocyte cells such that said selected population of cells hascharacteristics which are representative of the same cells taken from apatient suffering from a disease or disorder; (b) separating theleukocyte cells, which now include the activated or modified selectedpopulation of leukocyte cells, from the medium and washing the separatedcells; and (c) preserving the washed cells by lyophilization with anisotonic trehalose solution so that when they are reconstituted, thecells can be used an control cells in immunological assays includingflow cytometry assays.
 9. A process for preparing a mixture of preservedmammalian control cells useful in flow cytometry analysis in which aselected population of leukocyte cells in a normal leukocyte cellpopulation have been artificially activated or modified to expressantigenic characteristics which are expressed on the same leukocytecells in a person having a disease of disorder, comprising:(a)(i)separating the total leukocyte population in order to obtain a selectedpopulation of leukocyte cells and the remaining leukocyte cells; (ii)separately retaining both the selected population of leukocyte cells andthe remaining cells; (iii) artificially activating or modifying theselected population of leukocyte cells with an enzyme or chemicalreagent in a medium; (b)(1) separating the activated or modifiedpopulation of leukocyte cells from the medium and washing the same; and(ii) mixing the washed, activated or modified population of leukocytecells with the remaining leukocyte cells; or alternatively, mixing thewashed, activated or modified leukocyte cells with the remainingleukocyte cells and other non-activated or unmodified leukocyte cells,in an amount representative of a stage of the disease; and (c)preserving the washed cells by lyophilization with an isotonic trehalosesolution so that when they are reconstituted, the cells can be used ascontrol cells in immunological assays including flow cytometry assays.